The bio-degradable polymer poly (DL-lactide-co-glycolide) (PLG) has been used for many years by the pharmaceutical industry to deliver drugs and biologicals in microparticulate form in vivo. The United States FDA has recently approved a PLG microsphere 30-day delivery system for leuprolide acetate (Lupran Depot (registered trade mark)) to be used in the treatment of prostate cancer. A useful review of the potential of polymer microencapsulation technology for vaccine use is found in Vaccine, 1994, volume 12, number 1, pages 5-11, by William Morris et al.
As an alternative to encapsulation, it is also known to deliver antigens in phospholipid vesicles called liposomes, as described for example by Eppstein, D. A et al in Crit. Rev. Ther. Drug Carrier Syst. 1988, 5(2), pages 99-139. It is reported that a number of antigens have been delivered intraperitoneally using liposomes, including cholera toxin, malaria sporozoite protein and tetanus toxoid, and that influenza antigen has been delivered intra-nasally.
It is also known that, in certain circumstances, injection of naked DNA into tissue can lead to expression of a gene product coded by that DNA. For example, in 1984, work at the United States NIH reported that intrahepatic injection of naked, cloned plasmid DNA for squirrel hepatitis produced both viral infection and the formation of anti-viral antibodies in the squirrels.
WO-A-95/05853 describes methods, compositions and devices for administration of naked polynucleotides which encode biologically active peptides. This published application describes, inter alia, the injection of naked DNA coding for an immunogenic antigen with the aim of raising antibodies in the recipient of the naked DNA.
Liposomal delivery of DNA is also known, and is described, for example, in EP-A-0475178.
An alternative method for obtaining in vivo expression of a desired gene product is described in EP-A-0161640, in which mouse cells expressing bovine growth hormone are encapsulated and implanted into a cow to increase milk production therein.
EP-A-0248531 describes encapsulating linear poly (I:C) in microcapsules and using these to induce production of interferon.
WO-A-94/23738 purports to describe a microparticle containing DNA in combination with a conjugate that facilitates and targets cellular uptake of the DNA. In working examples, bombardment of cells by microparticles containing Tungsten is described. These examples appear little different to conventional bombardment of cells with DNA-coated metal particles. Furthermore, sonication is proposed in microparticle manufacture, a step that is known to risk DNA damage, and the presented data is inadequate and inappropriate to determine the integrity of the encapsulated DNA.
In the present invention, it is desired to deliver, in vivo, DNA that encodes proteins with immunogenic, enzymatic or other useful biological activity, usually under the control of an active eukaryotic promoter. Objects of the invention include improvement on vaccination therapies known in the art and improvement upon prior art gene therapy methods. Improvement of or alternatives to existing compositions and methods are desirable as these existing methods are known to contain a number of drawbacks.
WO-A-95/05853 describes administration of naked polynucleotides which code for desired gene products. However, the compositions and methods in this publication are suitable only for injection, requiring sterile procedures, and being in itself an unpleasant and awkward route of administration.
WO-A-94/23738 purports to provide a process in which encapsulated DNA is released from particles in the body of the recipient and then taken up by cells, although no accomplished in vivo examples are presented.
Morris W et al (`Potential of polymer microencapsulation technology for vaccine innovation`, Vaccine, Vol. 12, No. 1; pp5-11) is an article reviewing the PLG encapsulation field. It does not describe delivering DNA based vaccines. Instead, it describes delivering antigen based vaccines. Further, it only fleetingly describes preparation of microparticles that contain an internal component within a polymer shell. Instead, it primarily describes microparticles of the matrix type, that is to say within which an antigen is dispersed. Morris et al define such particles as "microspheres"--see page 5, column 2, lines 20-22--and the article deals extensively with such microspheres. Morris et al refer to a polymer shell that encapsulates an internal component as a "microcapsule".
The small section in the Morris paper on how to obtain microcapsules, from page 8, middle of column 2 to the top of column 1 on page 9 describes microcapsules of &gt;50 .mu.l in volume (about 1.2 mm in diameter).
Sah HK et al (`Biodegradeable microcapsules prepared by a w/o/w technique: effects of shear force to make a primary w/o emulsion on their morphology and protein release` J. Microencapsulation, Vol. 12, No. 1, 1995, pp 59-69) describes a water-in-oil-in-water method for the encapsulation of biologically active agents. The thrust of the article is on determining the influence of shear forces on the characteristics of the microcapsules obtained. We refer to the first two lines of the abstract. The size distribution of these particles was measured and found to be in the range 10-75 .mu.m. Further, Sah et al were not able to change the size of the microcapsules that they prepared. We refer again to the second paragraph in "Results and discussion" towards the end, where it is stated:
"However, in our experiments, the change of sheer rate from 11 to 23 krpm to produce the primary W/O emulsion did not result in a reduction in microcapsule size. No correlation between shear forces to make an initial W/O emulsion and the resulting microcapsule size was observed". PA1 (a) preparing an aqueous solution of DNA, said DNA comprising a sequence coding for a polypeptide in operative combination with at least a promoter sequence and optionally other sequences regulating or otherwise directing transcription of the DNA, said DNA being adapted to express the polypeptide in a mammalian recipient; PA1 (b) preparing a solution of polymer in an organic solvent; PA1 (c) forming an emulsion of the aqueous DNA solution in the organic polymer solution; PA1 (d) preparing an aqueous surfactant solution; PA1 (e) forming a double emulsion of (I) the emulsion from (c) in (II) the aqueous surfactant solution; PA1 (f) at elevated temperature, dispersing or otherwise removing the organic solvent so as to form microparticles of polymer having sizes up to 10 .mu.m in diameter and which contain said DNA; and PA1 (g) recovering the microparticles. PA1 providing a (water-in-oil)-in-water emulsion containing the DNA solution; and PA1 adding this emulsion to excess of a further aqueous phase to extract the oil phase and thereby form microparticles, PA1 wherein the aqueous solution of DNA comprises alcohol. PA1 (a) the antigens FHA, PT, 68 kd-Pertactin, tetanus toxin, gp48, NS1, Capsid, gp350, NS3, SA, I, NP E, M, gp340, F, H, HN, 35 kd protein, BP1, E1, E2, C, M, E and MSHA according to table 1; and PA1 (b) immunogenic fragments, variants and derivatives of the polypeptides of (a).
Many published patents and applications are in the name of the Southern Research Institute (SRI). In particular, U.S. Pat. No. 5,407,609 purports to describe in example 7, an emulsion based method for the manufacture of hollow particles. However, the methods detailed in U.S. Pat. No. 5,407,609 succeed in making relatively large particles, or at least particles over a wide range of sizes, where a significant portion of particles are larger than the biological activity cut off point of 10 microns. A large spread of particle sizes, such as that seen in U.S. Pat. No. 5,407,609 inevitably leads to much of the encapsulated agent being incorporated in particles of a size that are not appropriate for phagocytosis.